Figure 4

Effects of CT45 expression on self-renewal in endometrial cancer cell lines. To investigate of CT45 expression, stable knockdown of CT45 of IK-YBX2 cells and CT45 gene-rescued cells of them were established. (a) The level of CT45 expression of established cells was investigated by Western blotting. shRNAs significantly reduced the expression of CT45 (lane 3, 4 vs lane 5, 6), and the proteins were rescued using relevant gene expression (lane 7, 8 vs lane 9, 10). Full length blots are shown in Supplementary Fig. S5d online. (b) The serial spheres formation assays were performed using two different clones of IK-YBX2-shCT45 and IK-YBX2-sh control cells respectively. Representative spheres generated from a single cell after 14 days in non-adherent culture were shown (scale bar 150 μm). (c) Sphere formation was counted at passage (P) 1 to 5. The serial sphere forming capacity, was reduced in IK-YBX2-shCT45 cells compared with in IK-YBX2-sh control cells (**p < 0.01, *p < 0.05). (d) To exclude off target effect of shRNA, we also established cells which were rescued CT45 expression by introduction of CT45 cDNA into each clone of CT45 knocked down cells. Upon serial passage from P1-P4, IK-YBX2-shCT45-mock cells generated few tumorsphere during passages. Conversely, sphere formation efficiency was increased in IK-YBX2-shCT45-CT45 rescued cells and a significant increase was observed in of IK-YBX2 C3-shCT45-CT45 rescued cells (*p < 0.05). Data represent the means ± SD from three independent experiments.